Alternative nanomaterial and gene transfer solutions are needed to access the potential held in intracellular imaging, biopharmaceuticals, gene therapy and stem cell research. Delivery of small and macromolecules- including nucleic acids, drug molecules, imaging agents, peptides, antibodies and enzymes-into cells is critical to realizing their full potential in a rnge of research and therapeutic applications. Unfortunately, efficient intracellular delivery and transfection continue to elude researchers working with primary and stem cells in emerging application areas. OpenCell's vision is to create new intracellular delivery tools that enable researchers in emerging application areas to work with difficult-to-transfect cells for studies that are not currently possible. Our core technology, the Electrosonic Actuation Microarray (EAM), offers researchers a cost-effective and simple-to-use solution to uniform and scalable treatment of entire cell populations on a cell-by-cell basis. Multple Integrated Sample Treatment (MIST)-the subject of this project proposal-addresses cost and performance deficiencies of available methods by incorporating the innovative features of the EAM technology into a multichannel format. In addition, MIST uniquely streamlines protocol optimization and large-scale screening by evaluating treatment outcomes for multiple experimental conditions, including operating parameters and sample compositions, in parallel. During the Phase I project, a MIST prototype achieved biomolecule delivery performance equal to that of our single-sample STEAM system in a 4-channel format, without cross-contamination between channels. We have recently demonstrated advantages of our core technology for large molecule delivery (including in difficult-to- transfect primary monocytes) and for improving transfection outcomes with combined-mode treatment. It is anticipated that these advantages will carry over to the production MIST system. The primary objectives of this SBIR Phase II project are design, manufacture and characterization o a production-ready MIST instrument. To achieve these objectives, (1) MIST systems with stand-alone electronics and disposable multichannel cartridges for mechanoporation and DNA electrophoresis will be developed, and (2) transfection protocol optimization will be demonstrated, along with a comparison of relevant assessment criteria with our single-sample STEAM system and competing technologies.